Earticle

초록

영어

There are two types of glycosylation, N-linked and O-linked, respectively. N-linked glycan (or N-glycan) is attached to the asparagines (Asn), which is usually released by N-glycosylation specific enzyme known as peptide-N-glycosidase F (PNGase F). O-linked glycan (or O-glycan) is attached to the either serine (Ser) or threonine (Thr), however, there is no enzyme for the universal release of O-glycans from glycoproteins. In general, O-glycans are chemically released by β-elimination, which often release N-glycans that are abundant in human fluids. Thus, O-glycan release still remains analytical challenge in the field of glycomics. Here, we have developed the method to release O-glycans selectively and specifically. Mucin are large glycoproteins that carry dense clusters of O-linked glycans and IgG are glycoproteins that attacted only N-glycans. N and O mixed glycans were prepared in the lab to exist both N-glycans and O-glycans together in a sample. We approached three methods to compare and determine the best method to release selective and specific O-glycans release. 1) Method A : O-glycoproteins were eluted in acetonitrile (ACN) with 0.1% trifluoroacetic acid (TFA) from the cartridge that N-linked glycans were already removed. Glycoproteins were further treated with sodium borohydride (NaBH4) to release O-glycans and then enriched by porous graphitized carbon (PGC). We found that O-glycans were eluted in 20% ACN/H2O. 2) Method B : We separated N-glycans released by PNGase F and glycoproteins in mixed samples using molecular weight cut off (MWCO) 10,000 Da spin-column with centrifugation because glycoproteins molecular weight is higher than N-glycans. Glycoproteins were further treated with NaBH4 to release O-glycans and then enriched by PGC. We found that O-glycans were eluted in 20% ACN/H2O. 3) Method C : Glycoproteins in mixed samples after PNGase F treatment are precipitated by cold 100% ethanol. Glycoproteins were further treated with NaBH4 to release O-glycans and then enriched by PGC. We found that O-glycans were eluted in 20% ACN/H2O. Purified O-glycans were analyzed using Matrix-assisted Laser Desorption/Ionization time-of-flight mass spectrometry (MALDI-TOF MS). Indeed, we found O-glycans such as [Hex]1[HexNAc]2, [Hex]2[HexNAc]4 and [Hex]2[HexNAc]3[Fuc]1. This suggests that O-glycans can be selectively and specifically released under the absence of high abundant N-glycans using our approach. In the future, we will analyze enriched O-linked glycans quantitatively by nano-LC/chip Q-TOF MS to compare three methods. This analytical platform can be widely used for O-glycan release and enrichment.

저자

• Ha Neul Jeong [ Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon, Korea ]
• Myung Jin Oh [ Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon, Korea ]
• Serenus Hua [ Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon, Korea ]
• Hyun Joo An [ Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon, Korea ]

참고문헌

발행기관

간행물

표지보기 관심저널 등록

• 간행물명

  한국당과학회 학술대회

• 간기

  연간

• 수록기간

  2006~2022

• 십진분류

  KDC 517 DDC 614